Construction And Transformation Of RNAi Vector For Genes In Crossover Formation Of Brassica Napus

Recognition and pairing of homologous chromosome are essential to successful meiosis. Crossovers are formed between non-sister chromatids of homologous chromosomes, which cause the recombination of homologous chromosomes. Recombination increase genetic diversity and provide a material basis for natural selection. In recent years, research of meiosis in Arabidopsis thaliana and Oryza sativa show that there are many genes take part in the regulation of crossover, which make it possible for us to control the formation and frequency of crossover. Brassica napus(AACC), one of the most important oil crops in China, is an allopolyploid evolved from the hybrid of B. rapa(AA) and B. oleracea(CC). Because of the lack of natural and artificial mutants, there are fewreports on crossovercontrol in Brassica naups till now. In this study, we intend to down-regulate the expression of crossover-related genes in Brassica naups through RNAi technique and analysis the molecular mechanism of crossover formationin in Brassica napus. Moreover, we also want to introduce favorable genes of wild relatives into B. napus by sexual hybridization using those RNAi lines. Main results of this study are described as follows:1) We got RNAi target fragments of FIGL1, MHF1, XRCC2, MUS81 and ASY1 by RT-PCR from Brassica napus “westar” and constructed successfully each RNAi vector using pFGC5941 as framework.2) RNAi vector was introduced into Brassica napus “westar”, “J9709” and “JW76” by agrobacterium tumefaciens-mediate transformation method. After selection and cultivation, 78 regenerated plants had been obtained.3) PCR detectionidentified 48 transgenic positive plants in 66 regenerated plants, included 25, 8 and 12 plantswith FIGL1, MHF1 and XRCC2 transformation respectively. The average transformation frequency is 72.73%. Moreover, 12 plants with MUS81 transformation have been regeneratedbut without PCR detection. Unfortunately, there is no plant with ASY1 transformation were regenerated.4) Primers were designed according to conserved sequence of each gene. qRT-PCR analysis showed that there are 14 transgenic positive plants had lower gene expression level than those in wild typeplants in flower buds.5) No obvious difference of chromosome behaviors has been found in meiosis of transgenic positive plantscompared to wild type plants. This result isconsit with that a normal meiosis process but crossover frequency is increased in corresponding mutants in Arabidopsis thaliana.